The geneSex-lethal (Sxl) plays a pivotal role inDrosophila sexual development. Once activated in response to the X: A ratio signal in XX embryos,Sxl participates in appropriate implementation of all known aspects of sexual differentiation. We have attempted to identify new X-linked genes involved in sex determination, especially those involved in the regulation ofSxl. Since misregulation ofSxl, or that of the genes that regulate it, leads to female-specific lethality, or synergistic female-lethal gene interactions, or both, we used these criteria to screen about 10,000 EMS-treated chromosomes for (i) recessive female-specific lethality or (ii) enhanced female lethality intransheterozygous combination withSxl. Four potentially useful mutations—Sxldlf, fl-35, fl-46, 1–43—were recovered and a few of their properties were characterized. Approximate map positions of these mutations were determined by meiotic mapping. To understand their probable position(s) in the hierarchy of genes regulating sex determination, we studied dose-dependent interactions between them and mutations in genes known to affect sex determination by generating double and triple heterozygotes. These studies suggest that (i)Sxldlf is not defective in the ‘early’ regulation or functions ofSxl, and (ii)fl-35, fl-46 and1–43 are unlikely to be a part of the X: A ratio signal, i.e. they are not needed for the transcriptional activation ofSxl. On the other hand, they could be affecting post-transcriptional processing ofSxl transcripts.